Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these refernces may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Streptavidin, a protein produced by STreptomyces avidinii, forms a very strong and specific non-covalent complex with the water-soluble vitamin biotin. Streptavidin was discovered in 1963 (1) as part of an antibiotic system in culture filtrates of several species of Streptomyces. Later Chaiet and Wolf (2) established its chemical nature and determined its amino acid composition. Streptavidin is a nearly neutral 60,000 dalton protein. It consists of 4 identical subunits each having an approximate molecular weight of 15,000 daltons. Streptavidin binds 4 molecules of biotin per molecule of protein, and it is free of carbohydrate. Avidin, a basic glycoprotein usually isolated from chicken egg-whites, shares with streptavidin some common characteristics such as molecular weight, subunit composition and capacity to bind biotin and forming a complex with biotin of very high affinity (K.sub.D =10.sup.-15) (3-4). Streptavidin and avidin have different amino acid compositions, but both have an unusually high content of threonine and tryptophan. Although streptavidin and avidin (derived from egg-white) bind biotin with equally high affinity, streptavidin has the advantage of avoiding much of the undesirable, nonspecific binding associated with avidin at physiological pH. The reasons for this are: (1) the isoelectric point of streptavidin is close to neutral, that of avidin is 10 (thus avidin is positively charged at pH 7.0); and (2) streptavidIn contains no carbohydrate, while avidin contains approximately 7% carbohydrate.
At present, commercial preparations of streptavidin made by growing S. avidinii have several disadvantages: they are high in cost and are frequently contaminated with biotin, and, as a result do not have all four valences free for binding biotin. Furthermore, production of streptavidin from S. avidinii yields only limited quantities of streptavidin.
The present invention overcomes the disadvantages of present commercial preparations of streptavidin by providing an inexpensive source of streptavidin, which is essentially free of biotin contamination, and has all four valences free for biotin binding. The present invention contemplates vectors which can produce streptavidin in large quantities. Furthermore, improved streptavidins may be produced by site-directed mutagenesis.
There have been attempts in the past to devise methods for labeling and detecting small amounts of interesting proteins within living cells. Past methods have included fusing to genes encoding the interesting proteins a prokaryotic gene, e.g. the gene for beta-galactosidase. Expression of the resulting fused gene results in a fused polypeptide, e.g. one containing the amino acid sequence from beta-galactosidase which can be used for stabilization and isolation of the protein of interest. However, such methods could not be used to produce labeled proteins in vivo.
The present invention provides a method of generating labeled proteins in vivo, without the need for in vivo covalent chemical modification. The present method utilizes a marker protein which may be non-covalently attached to a tag which remains with the protein. This method may be used to produce labeled proteins in vivo or to isolate target proteins knowing only the structure of the gene which encodes them.
Biotin may be conjugated to a variety of biological molecules using the strong, specific biotin binding capacity of avidin or streptavidin. The fused gene of the present invention thus permits the detection, localization or purification of proteins, carbohydrates and nucleic acids.